Download citrate synthase (ec 4

1
Composed for Protocol Collection @ OXPHOS International.
Contributed by Dr. .
Title of the Protocol
Modified after:
.
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Reagents:
 Prepare 0.1 M Tris¯HCl, pH 8.0;
 Prepare 1 mM DTNB solution, 4 mg of 5,5'-dithiobis-(2-nitrobenzoic acid) per10 ml of 0.1
M Tris-HCl (pH 8.0) (fresh daily);
Procedure:
 In a total volume of 1 ml, the assay medium is composed of 0.1 mM DTNB (100 l), 0.36 mM
acetyl-CoA (30 l), 0.05 - 0.15 mg of mitochondrial protein, and 0.5 mM OAA (50 l), in Tris-HCL
buffer (~0.8 ml).
 Perform assay at 25°C;
 Set spectrophotometer to 412 nm, blank by air;
 Dissolve (mitochondria) sample in 0.1% Triton X-100;
 Add mitochondria to the mixture of DTNB and Acetyl-CoA, record absorbance changes for 1 min;
 Add 50 l OAA, record absorbance increase for 2 min;
 Calculate specific activity as mU/mg of mitochondrial protein; for that, subtract the rate of
absorbance changes before OAA addition (if any) from that after OAA addition and divide it by the
extinction coefficient for TNB, EmM=14.15 mM-1 cm-1 and by mitochondria protein content. 1 mU of
enzyme catalyzes the reduction of 1 nmol of DTNB per min.
Comments:
Supplement:
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